Anti klotho

Fixed samples were decalcified in 20% EDTA (pH 7.5), embedded in paraffin and cut into 5 μm sections using an HM360 microtome (Microm). Hematoxylin (VWR) and eosin (Sigma–Aldrich) staining was performed to assess histomorphology. Tartrate-resistant acid phosphatase (TRAP) (Sigma) was used to detect osteoclasts according to the manufacturers’ protocols. For immunofluorescence staining, slides were subjected to sodium citrate buffer at 95 °C for 20 min for antigen retrieval, permeabilized with 0.5% Triton X-100 (Beyotime) for 10 min, and blocked with 5% BSA for 1 h. Slides were incubated with Anti-Runx2 (1:200, Abcam, ab23981), Anti-GFP (1:50, Santa Cruz, sc-9996), Anti-RFP (1:50, Santa Cruz, sc-390909), Anti-Klotho (1:100, R&D, AF1819), Anti-Klotho (1:100, Santa Cruz, sc-515939), or Anti-Rankl (1:100, R&D, AF462) antibody overnight at 4 °C, and a fluorescence-conjugated secondary antibody, Alexa Fluor 488 or 568 (Invitrogen, 1:1000) for 1 h at room temperature. Nuclei were counterstained with DAPI (Vector). Images were captured on an Olympus confocal microscope FV3000 (Olympus).

Slides of HK-2 cells were fixed with 4% paraformaldehyde for 20 min and processed by a combined immunofluorescence and FISH protocol, which was developed for the simultaneous detection of Klotho and NEAT1^{22 (link)}. First, the slides were hybridized with 8 ng/μl NEAT1 probes (Ribobio, Guangzhou, China) at 37 °C overnight. Subsequently, the slides were incubated with anti-Klotho (1:25, Santa Cruz, USA) at 4 °C overnight. Then, the reaction was developed with FITC goat anti-mouse IgG (1:100, ProteinTech, China) for 1 h. Finally, nuclei were counterstained with DAPI. The slides were visualized for immunofluorescence and FISH with a fluorescence microscope at a magnification of ×400.

For Western blot analysis, 100 μg of total kidney protein were separated on SDS-polyacrylamide minigels by electrophoresis (21 (link)). After transfer by electroelution to PVDF membranes (GE Healthcare Limited, Little Chalfont, UK), blots were blocked for 1 h with 5% non-fat milk in Tris-buffered saline solution. Blots were then incubated overnight with primary antibodies for anti-VDR and anti-Klotho (1:500 for both; Santa Cruz Biotechnology, Santa Cruz, CA). The labeling was visualized with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit, 1:2,000, or anti-goat, 1:10,000; Sigma Chemical, St. Louis, MO) and enhanced chemiluminescence (ECL) detection (GE Healthcare Limited, Little Chalfont, UK). Kidney protein levels were further analyzed with a gel documentation system (Alliance 4.2; Uvitec, Cambridge, UK) and the software Image J for Windows (Image J-NIH Image). We used densitometry to quantitatively analyze the protein levels, normalizing the bands to β-actin expression (anti-β-actin, Sigma Chemical, St. Louis, MO).